Fasciola gigantica, F. hepatica and Fasciola intermediate ...
Isolation of Fasciola hepaticaTegumentAntigens · Fasciola hepatica tegument antigens wereisolated...
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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1980, p. 695-699 0095-1137/80/1 1-0695/05$02.00/0 Vol. 12, No. 5 Isolation of Fasciola hepatica Tegument Antigens GEORGE V. HILLYER Laboratory of Parasite Immunology, Department of Biology, University of Puerto Rico, Rio Piedras, Puerto Rico 00931 Fasciola hepatica tegument antigens were isolated from intact worms in the cold by using Nonidet P-40. Proof of the tegumental nature of the antigens was shown by the peroxidase-antiperoxidase immunocytochemical technique at the light microscope level. The potential of F. hepatica tegument antigens for the immunodiagnosis of rabbit and human fascioliasis was shown by Ouchterlony immunodiffusion, although cross-reactivity was evident in one of six serum samples from patients infected with Schistosoma mansoni. A genus-specific Fasciola antigen was found in F. hepatica tegument. Finally, F. hepatica tegu- ment contained antigens which protected mice from challenge infection with S. mansoni. Most procedures used in the extraction of parasite antigens for serodiagnosis begin by the breaking up of parasite components. These com- plex mixtures are then applied to the serological test being used. Parasitic trematodes have a glycocalyx-tegument complex which continu- ously secretes macromolecules. For example, Threadgold (10) found that the glycocalyx of the Fasciola hepatica tegument consists of two parts, an inner continuous layer tightly bound to the apical plasma membrane and an outer fi- brillar layer. Both layers are anionic and carbo- hydrate rich and thus contain glycoproteins and sialic acids. Wilson and Barnes (11) found that the plant lectin concanavalin A bound to the glycocalyx (membranocalyx) of Schistosoma mansoni: this surface coat had a half-life of 2 to 3 h. Thus glycocalyx antigens are probably con- tinuously secreted by the parasite, stimulating host response. It would be expected, then, that isolation of tegument antigens would be a simple approach to the collection of antigens which may be useful starting material for immuno- chemical purification of antigens which can then be applied to the immunodiagnosis of parasitic infections. In addition, since they are being shed by the parasite surface they may also be func- tional in protective immunity. This study concerns the isolation of F. hepa- tica tegument antigens (Fht) obtained from worms and their use in the serodiagnosis of experimental infections. MATERIALS AND METHODS Antigens. Bovine livers condemned by the U.S. Department of Agriculture because of fascioliasis were obtained at the slaughterhouse in Barrio Cubuy, Can- ovanas, P.R. The F. hepatica adult worms were re- moved from the bile ducts and washed repeatedly in ice-cold 0.01 M phosphate-buffered saline (PBS), pH 7.0. Groups of 10 worms were transferred to small beakers, covered with 25 ml of the nonionic detergent Nonidet P-40 (1% NP-40 in PBS), and incubated at either 4 or 37°C for 1 h. The NP-40 was obtained from BDH Chemicals Ltd., Poole, England. The fluid was then collected and centrifuged for 4 h at 50,000 x g (8°C). The supernatants were designated Fht (4°C) or Fht (37°C). Protein determinations were done by a micro-Lowry technique with bovine serum albumin as a standard and as described in Keleti and Lederer (7). The protein concentrations determined were as fol- lows: Fht (4°C), 925 ,gg/ml; Fht (37°C), 1,375 gg/ml. Samples of each antigen preparation were then con- centrated to 8 to 10 mg/ml by vacuum dialysis against PBS at 4°C. F. hepatica worms, obtained from the liver of a New Zealand white rabbit infected for 34 weeks, were washed as above. One worm was incubated either in 5 ml of PBS or in 5 ml of 1% NP-40 in PBS for i h at 37°C. The fluid from each was then centrifuged as above, and the supernatant was collected. The protein concentrations determined were as follows: PBS su- pernatant, 260 ug/ml; NP-40-PBS supernatant, 803 ,ug/ml. A sample of the NP-40-PBS supernatant was also concentrated to 8 to 10 mg/ml by vacuum dialysis. F. hepatica worm extracts were prepared as de- scribed previously by Hillyer (2) and used in Ouchter- lony immunodiffusion at a concentration of 16 mg (dry weight)/ml. Sera. Rabbits were infected with F. hepatica metacercariae obtained from laboratory-reared Lym- naea cubensis snails (kindly donated by Anne Frame and Pedro Bendezà, Inter-American University, San Juan Campus). They were bled repeatedly for the collection of serum for up to 20 weeks postinfection. Rabbits were hyperimmunized with F. hepatica crude worm extracts (bovine) for the collection of serum (anti-F. hepatica serum). A typical immuniza- tion protocol involved a first inoculation in five sub- cutaneous sites of a total of 10 mg (dry weight) of antigen emulsified in Freund complete adjuvant fol- 695 on June 29, 2020 by guest http://jcm.asm.org/ Downloaded from
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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1980, p. 695-6990095-1137/80/1 1-0695/05$02.00/0
Vol. 12, No. 5
Isolation of Fasciola hepatica Tegument AntigensGEORGE V. HILLYER
Laboratory of Parasite Immunology, Department of Biology, University of Puerto Rico, Rio Piedras,Puerto Rico 00931
Fasciola hepatica tegument antigens were isolated from intact worms in thecold by using Nonidet P-40. Proof of the tegumental nature of the antigens wasshown by the peroxidase-antiperoxidase immunocytochemical technique at thelight microscope level. The potential of F. hepatica tegument antigens for theimmunodiagnosis of rabbit and human fascioliasis was shown by Ouchterlonyimmunodiffusion, although cross-reactivity was evident in one of six serumsamples from patients infected with Schistosoma mansoni. A genus-specificFasciola antigen was found in F. hepatica tegument. Finally, F. hepatica tegu-ment contained antigens which protected mice from challenge infection with S.mansoni.
Most procedures used in the extraction ofparasite antigens for serodiagnosis begin by thebreaking up of parasite components. These com-plex mixtures are then applied to the serologicaltest being used. Parasitic trematodes have aglycocalyx-tegument complex which continu-ously secretes macromolecules. For example,Threadgold (10) found that the glycocalyx of theFasciola hepatica tegument consists of twoparts, an inner continuous layer tightly bound tothe apical plasma membrane and an outer fi-brillar layer. Both layers are anionic and carbo-hydrate rich and thus contain glycoproteins andsialic acids. Wilson and Barnes (11) found thatthe plant lectin concanavalin A bound to theglycocalyx (membranocalyx) of Schistosomamansoni: this surface coat had a half-life of 2 to3 h. Thus glycocalyx antigens are probably con-tinuously secreted by the parasite, stimulatinghost response. It would be expected, then, thatisolation of tegument antigens would be a simpleapproach to the collection of antigens whichmay be useful starting material for immuno-chemical purification of antigens which can thenbe applied to the immunodiagnosis of parasiticinfections. In addition, since they are being shedby the parasite surface they may also be func-tional in protective immunity.This study concerns the isolation of F. hepa-
tica tegument antigens (Fht) obtained fromworms and their use in the serodiagnosis ofexperimental infections.
MATERIALS AND METHODSAntigens. Bovine livers condemned by the U.S.
Department of Agriculture because of fascioliasis wereobtained at the slaughterhouse in Barrio Cubuy, Can-ovanas, P.R. The F. hepatica adult worms were re-moved from the bile ducts and washed repeatedly in
ice-cold 0.01 M phosphate-buffered saline (PBS), pH7.0. Groups of 10 worms were transferred to smallbeakers, covered with 25 ml of the nonionic detergentNonidet P-40 (1% NP-40 in PBS), and incubated ateither 4 or 37°C for 1 h. The NP-40 was obtained fromBDH Chemicals Ltd., Poole, England. The fluid wasthen collected and centrifuged for 4 h at 50,000 x g(8°C). The supernatants were designated Fht (4°C) orFht (37°C). Protein determinations were done by amicro-Lowry technique with bovine serum albumin asa standard and as described in Keleti and Lederer (7).The protein concentrations determined were as fol-lows: Fht (4°C), 925 ,gg/ml; Fht (37°C), 1,375 gg/ml.Samples of each antigen preparation were then con-centrated to 8 to 10 mg/ml by vacuum dialysis againstPBS at 4°C.
F. hepatica worms, obtained from the liver of aNew Zealand white rabbit infected for 34 weeks, werewashed as above. One worm was incubated either in5 ml of PBS or in 5 ml of 1% NP-40 in PBS for i h at37°C. The fluid from each was then centrifuged asabove, and the supernatant was collected. The proteinconcentrations determined were as follows: PBS su-pernatant, 260 ug/ml; NP-40-PBS supernatant, 803,ug/ml. A sample of the NP-40-PBS supernatant wasalso concentrated to 8 to 10 mg/ml by vacuum dialysis.
F. hepatica worm extracts were prepared as de-scribed previously by Hillyer (2) and used in Ouchter-lony immunodiffusion at a concentration of 16 mg (dryweight)/ml.
Sera. Rabbits were infected with F. hepaticametacercariae obtained from laboratory-reared Lym-naea cubensis snails (kindly donated by Anne Frameand Pedro Bendezà, Inter-American University, SanJuan Campus). They were bled repeatedly for thecollection of serum for up to 20 weeks postinfection.
Rabbits were hyperimmunized with F. hepaticacrude worm extracts (bovine) for the collection ofserum (anti-F. hepatica serum). A typical immuniza-tion protocol involved a first inoculation in five sub-cutaneous sites of a total of 10 mg (dry weight) ofantigen emulsified in Freund complete adjuvant fol-
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lowed by four or more inoculations, each with 5 mg ofantigen in Freund incomplete adjuvant.
Rabbits were immunized three times each with 1mg of protein of Fht (bovine) per ml obtained asdescribed above at 40C (anti-F. hepatica tegumentserum). The first inoculation (five sites) was done withan equal volume (1 ml) of Freund complete adjuvant,and the subsequent inoculations were done withFreund incomplete adjuvant. Bleedings were done 1week after the final inoculation for the collection ofserum.
Rabbit antiserum to F. hepatica arc 2, which isgenus specific (9), was kindly donated by André Cap-ron.
Rabbit antisera were also prepared to several F.hepatica antigen preparations which have been shownto protect mice from challenge infections with S. man-soni cercariae (summarized in references 4and 6). These include F. hepatica antigens whichbind to concanavalin A-Sepharose 4B, denotedFhConA (El-i), and a purified F. hepatica antigen whichcross-reacts with S. mansoni, denoted Fh5s 11(M) (5).Human sera were obtained from six individuals from
France with confirmed fascioliasis (sera kindly do-nated by André Capron), from six individuals fromEgypt with medium (300 eggs per g) to high (1,680eggs per g) excretion levels of S. mansoni (sera kindlydonated by M. A. El Alamy), and six individuals fromPuerto Rico with no detectable intestinal parasites.
Serological tests. Ouchterlony immunodiffusionwas done with 1% agarose as described previously (2)either on glass plates or on Gel Bond (FMC Corp.,Marine Colloids Division, Rockland, Maine).
Peroxidase-antiperoxidase immunocytochem-ical technique. At 21 days postinfection, F. hepaticaworms were removed from mouse livers, fixed 15 minin 3% paraformaldehyde in 0.1 M phosphate buffer(pH 7.3), and then placed in phosphate buffer. Wormswere dehydrated in ethanol, embedded in paraffin,and sectioned at 6 to 10 ,um. Sections were deparaffm-ized in xylene, air dried, and then exposed to theperoxidase-antiperoxidase technique of Sternberger etal. (8). Primary rabbit serum against Fh, was used ata 1:1,000 dilution. Sections were not additionallystained.
this was only 32% of that obtained under similarconditions when 1% NP-40 was added. Whenthese antigens were reacted by Ouchterlony im-munodiffusion against a rabbit anti-F. hepaticawhole worm serum the complexity of the systembecame apparent. Many of these tegument an-tigens are logically also found in the F. hepaticawhole worm extracts (Fig. 1). This also suggeststhat the important immunodiagnostie antigens
FhWWE
aF
FhpBS
Fht8
i,çi1
Fht0.8
FIG. 1. Ouchterlony immunodiffusion comparinga rabbit serum to F. hepatica crude worm extract(aFh) with various antigen preparations of F. hepa-tica (see text). FhWWE is a crude worm extract. Fh,were obtained by incubation with 1% NP-40 and wereof bovine origin; the preparation was at a proteinconcentration of8 or 0.8 mg/ml. FhpBs were obtainedin the cold after incubation with PBS; the antigenconcentration ofFhpBs was 260pg/ml. Note that mostFhNP-o linked with FhIWWE, indicating identity.
FhWWEe
Fht8.S
RESULTS AND DISCUSSIONF. hepatica worms (bovine) incubated in NP-
40 at 37°C shed approximately 1.5 times moreLowry-reactive material than those incubated at4°C. When the protein concentrations were ad-justed and both antigen preparations were com-pared by Ouchterlony immunodiffusion againstthe serum from a rabbit immunized with F.hepatica worm extracts or a rabbit infected withF. hepatica for 12 weeks, comparable lines ofprecipitation were seen. Since incubation at37°C may result in proteolysis due to activationof enzymes, isolation of Fht was subsequentlydone only at 4°C.
F. hepatica worms (rabbit) incubated in PBSat 37°C shed Lowry-reactive material, although
R Fh
FhPBS Fht0.8
FIG. 2. Same conditions as in Fig. 1, except thatthe serum was obtained from a rabbit infected withF. hepatica (RFh) for 12 weeks. Note that two majorantigens are seen where Fht were used at a concen-tration of8 mg/ml; a third antigen s best seen whereFht were diluted 10-fold.
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FhWWE10
20 12/
i18 14
16 aFharc 2 aFhtFIG. 3. Ouchterlony immunodiffusion ofthe serum
from a rabbit infected with F. hepatica for 10 to 20weeks (outer wells) reacted with Fht at a concentra-tion of 8 mg/ml. This rabbit was first positive to Fhtat 4, but not 2, weeks postinfection.
j''
0ab"
.b
'FIi
.V
-; e*-i,
c
v
FIG. 4. Ouchterlony immunodiffusion of the sera
from six patients (a to f) with confirmed fascioliasisreacted against Fht (center well) at a concentrationof 8 mg/ml. Note the presence of at least three differ-ent antigen-antibody systems in serum c.
of crude F. hepatica are also found on thetegument. The Lowry-reactive material ob-tained by incubating the worms in PBS atoneappeared poorly reactive although this may bea factor of concentration. When these antigenswere reacted with the serum from the rabbitinfected with F. hepatica for 12 weeks, a muchsimpler pattern was obtained (Fig. 2). Thus, 2major Fht were apparent at 8 mg/ml, and a thirdwas clearly evident when the antigen was diluted10-fold.
It was then of interest to see whether Fht wereuseful as a preparation for the immunodiagnosisof rabbit fascioliasis. Sera from rabbits infected
FIG. 5. Ouchterlony immunodiffusion of F. hepa-tica adult worm extract (FhWWE) reacted against arabbit anti-Fht serum (aFht) and a rabbit serumcontaining specific antibody to Fasciola arc 2(aFh",. 2) (see text). Note that the arc 2 antibody linkswith one Fht antibody, indicating identity.
2
FIG. 6. Reactivity of three different rabbit seraagainst Fht by Ouchterlony immunodiffusion. SerumI contained a pure antibody prepared from F. hepa-tica which cross-reacted with S. mansoni; serum 2contained antibodies to F. hepatica antigens whichbound to concanavalin A-Sepharose 4B and wereeluted with a-methylglucopyranoside; serum 3 con-tained antibodies to F. hepatica adult worm extracts.Note linkage of a common antibody in all three sera.The antigens used to prepare each of the sera havebeen shown to protect mice from challenge infectionwith S. mansoni.
with F. hepatica were positive by 4 (but not 2)weeks of infection when reacted with 8 mg ofFht (bovine) per ml. A typical rabbit antibodyresponse is seen in Fig. 3, which shows theprecipitin patterns from 10 to 20 weeks of infec-tion. As many of three lines of precipitation wereclearly seen at 14 weeks.That Fht may be useful for the immunodi-
agnosis of human fascioliasis is also evident. Allsix cases of human fascioliasis gave at least onefine of precipitation against Fht. At least threeantigen-antibody systems were evident (Fig. 4,
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FIG.. 7. Peroxidase-antiperoxidase immunocytochemical method reacting a rabbit anti-Fht serum with asection of an F. hepatica worm obtained from a mouse infected for 21 days. Note that the dark grains areuneven and only appear on the wormn surface. The bar represents 20 p.m.serum c.) None of the normal controls werepositive against Fht. However, one of the six S.mansoni cases did react with Fht. Thus, thesource of this cross-reactivity must be identifiedand absorbed out to increase the specificity ofthe antigen preparation.
F. hepatica worm extracts have been shownto comprise a complex mosaic of antigens, andmany of these have been enumerated by im-munoelectrophoresis (1). The one denoted arc 2has been shown to be genus specific (9). Theanti-Fht serum was compared with an anti-Fas-ciola arc 2 serum by Ouchterlony immunodif-fusion by reacting both with a crude F. hepaticaworm extract. The Fasciola-specific antibodylinked with one of the Fht antibodies, indicatingidentity (Fig. 5). Thus, Fasciola arc 2 is clearlya tegument antigen.
F. hepatica antigens have been shown to pro-tect mice from challenge infection with S. man-soni cercariae (reviewed in references 4 and 6).Three antisera against different F. hepatica an-tigen preparations which induce protectionagainst challenge with S. mansoni all reactedwith Fht (Fig. 6). Significantly, one of the anti-bodies was common to the three sera (5). This
suggests that the F. hepatica antigens whichprotect against S. mansoni are tegument anti-gens. I have shown that infection with F. hepa-tica also protects mice from challenge with S.mansoni (3). Thus, it is possible that the mech-anism of protection may in part be due to theshedding of the Fht.
Finally, the method of isolation of Fht usingdetergents at cold temperatures suggested thatonly surface (tegument) antigens were being iso-lated. With the peroxidase-antiperoxidase im-munocytochemical technique (8), an anti-Fht se-rum was reacted with sections of F. hepaticaworms isolated from infected mice. The locali-zation was exclusively on the worm surface (Fig.7). Thus, Fht are truly tegument antigens.
Additional analysis of these antigens is war-ranted both for the improvement ofserodiagnos-tic tests for fascioliasis and for studies on im-munity to schistosomes. These studies are cur-rently under way.
ACKNOWLEDGMENTSThese studies were supported by Public Health Service
grant no. RR-8102-08 from the National Institutes of Healthand administered by the Division of Research Resources, theEdna McConnell Clark Foundation, and the University of
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Puerto Rico Office of Graduate Studies and Research.Special thanks to Elliot Kieff for suggesting the initiation
of this study. The technical assistance of Zulma Sânchez deGavilanes and Richard Demaree is gratefully acknowledged.
LITERATURE CITED1. Biguet, J., A. Capron, and P. Tran Van Ky. 1962. Les
antigenes de Fasciola hepatica. Etude electrophore-tique et immunoelectrophoretique. Identification desfractions et comparison avec les antigenes correspon-dant a sept autres helminthes. Ann. Parasitol. Hum.Comp. 37:221-231.
2. Hillyer, G. V. 1975. Use of counterelectrophoresis todetect infections of Fasciola hepatica. J. Parasitol. 61:557-559.
3. Hillyer, G. V. 1976. Can we vaccinate against schisto-somes? Fed. Proc. 35:2568-2571.
4. Hillyer, G. V. 1979. Schistosoma mansoni: reduced wormburdens in mice immunized with isolated Fasciola he-patica antigens. Exp. Parasitol. 48:287-295.
5. Hillyer, G. V., and M. Cervoni. 1978. Laurell crossedimmunoelectrophoresis and affinity chromatographyfor the purification of a parasite antigen. J. Immunol.Methods 20:385-390.
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6. Hillyer, G. V., and L. Sagramososo de Ateca. 1979.Immunity in Schistosoma mansoni using antigens ofFasciola hepatica isolated by concanavalin A affinitychromatography. Infect. Immun. 26:802-807.
7. Keleti, G., and W. H. Lederer. 1974. Handbook ofmicromethods for the biological sciences. Van NostrandReinhold Co., New York.
8. Sternberger, L. A., P. H. Hardy, Jr., J. J. Cuculis,and H. G. Meyer. 1970. The unlabeled antibody en-zyme method of immunohistochemistry. Preparationand properties of soluble antigen-antibody complex(horseradish peroxidase-antihorseradish peroxidase)and its use in identification of spirochaetes. J. Histo-chem. Cytochem. 18:315-333.
9. Tailliez, R., R. Mangalo, and S. Korach. 1967. Isole-ment d'un antigen spécifique de la grande douve du foie(Fasciola hepatica L.). C.R. Acad. Sci. Ser. D. 265:466-469.
10. Threadgold, L. T. 1976. Fasciola hepatica: ultrastruc-ture and histochemistry of the glycocalyx of the tegu-ment. Exp. Parasitol. 39:119-134.
11. Wilson, R. A., and P. E. Barnes. 1976. The formationand turnover of the membranocalyx on the tegument ofSchistosoma mansoni. Parasitology 74:61-71.
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